This invention relates in general to novel polynucleotides isolated from cDNA libraries of human fetal liver-spleen and fetal liver and to polypeptides encoded by these polynucleotides. In particular, the invention relates to a human chemokine receptor that is a member of a family of G protein-coupled receptors characterized by seven transmembrane domains.
It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve GTP-binding proteins (termed xe2x80x9cG proteinsxe2x80x9d) and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 351:353-354 (1991)). Examples include xe2x80x9cG protein-coupled receptors,xe2x80x9d such as rhodopsin and the receptors for the adrenergic ligands and dopamine (Kobilka, B. K., et al., PNAS 84:46-50 (1987); Kobilka, B. K., et al., Science 238:650-656 (1987); Bunzow, J. R. et al., Nature 336:783-787 (1988)); G proteins themselves; effector proteins, e.g., phospholipase C, adenylyl cyclase, and phosphodiesterase; and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M. I., et al., Science, 252:802-8 (1991)).
In the inactive state, the membrane-associated G protein is a heterotrimer of alpha, beta, and gamma subunits in which the alpha subunit is bound to GDP. The binding of a ligand to a G protein-coupled receptor stimulates a receptor-G protein interaction that results in the exchange of GDP for GTP on the alpha subunit. The alpha subunit then dissociates from the beta-gamma subunits and interacts with an effector. In the case of epinephrine, for example, a G protein couples the beta-adrenergic receptor to adenylyl cyclase, stimulating the production of cAMP. The resultant rise in cAMP levels activates the cAMP-dependent protein kinase A, which phosphorylates and activates glycogen phosphorylase kinase. The latter, in turn, phosphorylates glycogen phosphorylase, producing a characteristic hormone-stimulated increase in enzymatic activity. Hydrolysis of GTP to GDP, catalyzed by the G protein itself, returns the G protein to its basal, inactive form. Thus, the G protein serves a dual role in signal transduction, namely, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
G protein-coupled receptors mediate the actions of a wide variety of extracellular signals including light, odorants, peptide hormones, and neurotransmitters and have been identified in organisms as evolutionarily divergent as yeast and humans. (See generally, Dolman et al., Ann. Rev. Biochem. 60:653-699 (1991).) Many G protein-coupled receptors share a similar topological motif consisting of seven hydrophobic (and potentially alpha-helical) regions predicted to span the lipid bilayer. Indeed, the transmembrane domains of such receptors are generally the most highly conserved regions of the proteins. Structure-function analyses carried out on the xcex12-, xcex21-, and xcex22-adrenergic receptors have shown that the fourth transmembrane segment (TMS IV) contains domains that contribute to the binding selectivity of various agonists and antagonists. TMS VII was shown modulate binding to receptor antagonists. Domains responsible for G protein binding are found the region of the protein encompassing TMS V and TMS VI along with the connecting cytoplasmic loop. These studies also implicate the cytoplasmic loops near TMS""s V-VII as determinants of G protein coupling and specificity.
Many 7-TMS receptors include a number of conserved cysteine residues. In both rhodopsin and the xcex22-adrenergic receptor, cysteines located in the C-terminal domain distal to TMS VII are covalently modified by palmitoylation. The xcex22-adrenergic receptor has been shown to contain two disulfide bonds that are required for normal ligand binding. Site-directed mutagenesis has revealed that four cysteines are essential for proper cell surface expression and ligand binding. Similar studies of rhodopsin indicate that, as in the xcex22-adrenergic receptor, a disulfide bond in a hydrophilic loop is critical for directing and/or stabilizing interactions that form the ligand binding domain. By contrast, the yeast xcex1-factor receptor has only two cysteine residues, both of which may be replaced by site-directed mutagenesis without any adverse effect on receptor function.
7-TMS receptors also share the ability to become desensitized, a process by which the receptors become refractory to further stimulation after an initial response, despite the continued presence of the original stimulus. Desensitization results from a reduction in the number of cell surface receptors or from an attenuation of the interaction between the receptor and the G protein (i.e., receptor-G protein xe2x80x9cuncouplingxe2x80x9d). Many G protein-coupled receptors of the seven-transmembrane-segment class (hereafter xe2x80x9c7-TMS receptorsxe2x80x9d) are rapidly uncoupled after exposure to agonists, a process regulated, at least in part, by phosphorylation.
The 7-TMS receptor family includes receptors for members of the chemokine family of inflammatory cytokines, such as interleukin-8 (hereafter IL-8). The name xe2x80x9cchemokinexe2x80x9d is derived from the ability of these proteins to stimulate chemotaxis of leukocytes. Indeed, chemokines comprise the main attractants for inflammatory cells during inflammatory and immune responses. See generally, Baggiolini et al., Advances in Immunology, 55:97-179 (1994). Chemokines have been shown to recruit a wide range of leukocytes to sites of infection, inflammation, and disease. For example, chemokines have been shown to be directly involved in the inflammatory process associated with conditions such as allergies (J Clin Invest Oct. 1, 1997; 100(7):1657-1666 Teixeira MM et al.), asthma (J Immunol Nov. 1, 1997;159(9):4593-4601 Lamkhioued B, et al.), arthritis (J Exp Med Jul. 7, 1997;186(1):131-137 Gong J H et al.), gastric inflammation (Physiol Pharmacol September 1997; 48 (3):405-413 Watanabe N et al.), injury (Eur J Neurosci Jul. 9, 1997;9(7): 1422-1438 Bartholdi D, Schwab M E), transplantation rejection (Transplantation Jun. 27, 1997;63(12):1807-1812 Fairchild R L et al.) and autoimmune disorders (J Neuroimmunol July 1997;77(1):17-26 Miyagishi R et al).
Members of the chemokine family generally exhibit 20-70% amino acid identity to one another and contain several highly-conserved cysteine residues. Chemokines can be classified into various subclasses or subfamilies by virtue of the position and spacing of a set of conserved cysteines, designated C-X-C (e.g., IL-8), C-C (e.g., RANTES) and C (e.g., lymphotactin). The C-X-C subfamily has the first two conserved cysteines separated by one amino acid, and the genes encoding the C-X-C subfamily are predominantly located on human chromosome 4. The C-C subfamily has two adjacent cysteines, and the genes encoding the C-C subfamily are predominantly located on human chromosome 17. The C subfamily has one of the first two conserved cysteines, and the genes encoding the C subfamily are predominantly located on human chromosome 17.
C-X-C chemokines IL-8, GROxcex1, GROxcex2, GROxcex3, ENA-78, NAP-2, PF4, and xcex3IP10 form a subfamily of neutrophil chemoattractants. IL-8, GROxcex1, and NAP-2 have been shown to compete for similar binding sites on neutrophils and elicit similar biological effects. At least two IL-8 receptors have been characterized, and their genes have been mapped to chromosome 2q34-35. One, designated IL-8 receptor type I, has been shown to bind GROxcex1 and NAP-2, in addition to IL-8. IL-8 acts on neutrophils to induce chemotaxis, hydrogen peroxide production, and exocytosis of intracellular granules, which is associated with an increase in the number of certain neutrophil receptors, such as the CR3 (C3bi) adhesion receptor. IL-8 also induces chemotaxis in basophils and lymphocytes, T lymphocytes, in particular. GROxcex1 and NAP-2 elicit effects in neutrophils similar to those elicited by IL-8.
The invention is based on polynucleotides isolated from cDNA libraries prepared from human fetal liver-spleen and fetal liver. This polynucleotide encodes a novel chemokine receptor having the amino acid sequence shown in FIG. 2 (SEQ ID NO:3). The invention provides a polynucleotide including a nucleotide sequence that is substantially equivalent to this polynucleotide. Polynucleotides according to the invention can have at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide encoding a polypeptide including SEQ ID NO:3. The invention also provides the complement of the polynucleotide including a nucleotide sequence that has at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide encoding a polypeptide including SEQ ID NO:3. In one embodiment, the polynucleotide of the invention includes a polynucleotide encoding a polypeptide including SEQ ID NO:3. In a variation of this embodiment, the polynucleotide includes a polynucleotide selected from SEQ ID NOs: 1 and 2. The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA.
Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. In exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.
A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (cf Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotide sequences for joining to polypeptides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide. In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
An additional aspect of the invention is a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide. The polypeptide can be recovered from the culture, conveniently from the culture medium, and further purified.
The invention further provides a polypeptide including an amino acid sequence that is substantially equivalent to SEQ ID NO:3. Polypeptides according to the invention can has at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to SEQ ID NO:3. In one embodiment, the polypeptide includes the amino acid sequence of SEQ ID NO:3.
The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody which specifically binds the polypeptide. The polypeptides of the invention are also useful for identifying the presence of and/or purifying a member of the IL-8 subfamily of chemokines. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.
Another aspect of the invention is an antibody that specifically binds the polypeptide of the invention. Such antibodies can be either monoclonal or polyclonal antibodies, as well fragments thereof and humanized forms or fully human forms, such as those produced in transgenic animals. The invention further provides a hybridoma that produces an antibody according to the invention. Antibodies of the invention are useful for detection and/or purification of the polypeptides of the invention as well as diagnosis or therapy of activated or inflamed cells and/or tissues. In addition, the polypeptides and antibodies of the invention are useful in methods for preventing neutropenia, preventing inflammatory or other immune responses, and inhibiting disease states associated with the hyperproliferative states of progenitor cells.